(A, B) Induction of luciferase under control of the MMTV promoter in COS-7 cells by wild-type (WT) MR and MR with the phosphomimetic S843E mutation in the presence or absence of 1 nM aldosterone (A) or 100 nM cortisol (B). Luciferase activity is expressed as % of WT MR activity in presence of ligand. Aldo, aldosterone; Cort, cortisol. Mean ± SEM are shown; n = 6 independent assays in each group; **P < 0.01.(C) Localization of WT or mutant MR in nuclear and cytoplasmic fractions in presence of 1 nM aldosterone. α–MR detects no signal without expression of MR. Unlike MRWT, MRS843E is almost exclusively cytoplasmic.(D) Affinity of [3H]aldosterone for MR. MRWT and MRS843E were expressed in COS-7 cells, and the binding affinity for [3H]aldosterone and Scatchard analysis was performed. Each data point was analyzed in duplicate and the mean value is shown. MRS843E shows drastically reduced ligand binding. See also .

(A) Antibodies specific for MRS843-P (α-MRS843-P) were incubated with phosphorylated and non-phosphorylated MR peptides spotted on a nitrocellulose membrane, followed by staining with peroxidase-conjugated donkey anti-rabbit antibody. The results demonstrate specificity for the phosphopeptide.(B) Western blot of COS-7 cell lysates expressing human MR using antibodies to total MR or MRS843-P. Samples were loaded in duplicate. α-MRS843-P specifically recognizes a protein the size of MR.(C) Phosphatase treatment eliminates α-MRS843-P signal. FLAG-tagged human MR was purified, and incubated with or without calf intestinal alkaline phosphatase (CIP) and analyzed by Western blotting (see also ).(D) Expression of MRS843-P in kidney. Total kidney lysates were prepared from mice fed normal chow and subjected to Western blotting with α-MRS843-P. The results from two mice are shown.(E) Specificity of α-MRS843-P in kidney lysates. Western blots of total kidney lysates from WT mice were stained with α-MRS843-P without or with CIP treatment then without peptide competition, with competition using the non-phosphorylated version of the immunizing MR peptide, then competition with the immunizing MR phosphopeptide. Phosphatase treatment or competition with immunizing phosphopeptide eliminates the antibody signal.(F) Mouse kidney lysates were fractionated into nuclear and cytoplasmic fractions and stained with α-MR and α-MRS843-P antibodies. Total MR is predominantly nuclear; MRS843-P is confined to the cytoplasm.(G) COS-7 cells expressing MR-GFP were stained with α-MRS843-P antibody (red) and DAPI (blue) 1 hr after the addition of 1 nM aldosterone. Arrows indicate cells expressing MR-GFP, which were detected by fluorescence microscopy. MR-GFP was mainly nuclear with less signal in the cytoplasm. In contrast, MRS843-P was virtually exclusively cytoplasmic. No MRS843-P signal was detected in cells that did not express MR-GFP (asterisks). Scale bar represents 10 μm.(H) WT mouse kidney sections were stained for α-MRS843-P (green) or AQP2 (a marker of principal cells in the CD, red). MRS843-P is cytoplasmic and only seen in AQP2-negative cells (intercalated cells).(I) Kidney sections were co-stained for MRS843-P (green) and AE1 (red), which is expressed on the basolateral membrane of α-intercalated cells. MRS843-P is found in AE1-positive cells (arrows) and also in some AE1-negative cells (asterisks, which are inferred to be β-intercalated cells; confirmed in ).(J) Kidney sections stained for α-MR (red) and α-MRS843-P (green). Total MR is predominantly nuclear with half the cells (inferred to be intercalated cells) also showing cytoplasmic staining for MRS843-P. Scale bars represent 50 μm in (H-J). See also .

(A) Perfusion-fixed, WT kidney sections were stained with α-WNK4 (green) or AQP2 (red). WNK4 is present in both AQP2-positive principal cells and in AQP2-negative intercalated cells (arrows; see also ). Scale bar represents 50 μm.(B) MRS843-P levels in kidneys of TgWNK4Q562E mice carrying PHAII mutant WNK4 (Q562E) and WT mice. Blots show biological replicates. Bar graphs show results of quantitation (n = 4). Data expressed as mean ± SEM; *P < 0.05.(C and D) Effects of aldosterone infusion on the levels of pendrin and B1 H+ ATPase in membrane fractions of kidneys from WT and TgWNK4Q562E mice. Bar graphs in (D) show the results quantitation in aldosterone-treated animals compared to controls (n = 6). Data expressed as mean ± SEM.(E) Parallel changes in levels of MRS843-P, pendrin and B1 H+ ATPase in response to physiologic and genetic manipulations. Levels of MRS843-P are inversely related to levels of B1 H+ ATPase and pendrin; the increase in pendrin and B1 levels with dephosphorylated receptor is dependent upon aldosterone. Red indicates increased and green indicates decreased levels.(F) Correlation of MRS843-P and pendrin or B1 H+ ATPase levels. Each data point compares the response of MRS843-P and either pendrin (left) or B1 H+ ATPase (right) in each physiologic or genetic manipulation described in (E) (comparisons of Slc12a3−/− + MRA to Slc12a3−/−, and of Tg-WNK4Q562E to WT (in which aldosterone levels are not increased) are not included). For MRS843-P levels in Tg-WNK4Q562E + aldosterone group, levels in WT mice are used as reference. See also .

Physiologic role of MRS843-P in response to hyperkalemia and volume depletion

(A) Mechanism for MR-dependent regulation of pendrin and B1 H+ ATPase in intercalated cells. High K+ increases MRS843-P, which prevents activation of MR by ligand. Dephosphorylation of MRS843-P in response to AII signaling restores receptor competence and upregulation of pendrin and B1 H+ ATPase in response to aldosterone.(B) The major apical electrolyte flux mediators seen in the three major cells types of the CNT and CD of the distal nephron are shown. MR is present in all three; however, MRS843-P (denoted ‘P’) is only seen in intercalated cells.(C) In hyperkalemia, aldosterone binds and activates MR in principal cells, but not intercalated cells. This increases electrogenic Na+ reabsorption via ENaC and provides the lumen-negative potential required for K+ secretion.(D) In volume depletion, in addition to MR signaling in principal cells, AII signaling reduces MRS843-P, which results in MR signaling in intercalated cells. This results in Cl− reabsorption via the combined activities of the apical H+ ATPase and the apical Cl−/HCO3− exchanger Pendrin. Increased Cl− reabsorption neutralizes the lumen-negative potential, preventing increased K+ efflux. AGTR1, AII type 1 receptor. See also .